Identification Of Naturally Occurring Inhibitors Against Human Carboxylesterase 2 In White Mulberry Root-bark

Purpose: White Mulberry Root-bark(WMR)is an edible Chinese herbal used for the treatment of inflammation,nephritis and asthma.Irinotecan(CPT-11),a widely used anticancer agent,can be hydrolyzed by h CE2 and then release its active metabolite which caused CPT-11 associated life-threatening diarrhea.Carboxylesterase 2(h CE2)plays an important role in oral bioavailability of prodrugs.Co-administration with potent h CE2 inhibitors can minimize CPT-11 associated life-threatening diarrhea in patients,and then improve the patient’s quality of life.This study aimed to investigate the inhibitory effects of ethanol extract from WMR against human carboxylesterase 2(h CE2),as well as to identity and character natural h CE2 inhibitors in this herbal,which may provide theoretical basis for WMR minimizing Irinotecan associated life-threatening diarrhea.Means: 1.Fluorescein Diacetate(FD)was used for rapid screening of the inhibitory effects of WMR extract towards h CE2.FD and its hydrolytic metabolite were analyzed by Synergy H1 Multi-Mode Reader.2.Chemical fingerprinting and fraction collection were performed on a Shimadzu Prominence UFLCTMsystem.The LC fractions were collected consecutively at 30 second intervals.The inhibition profile of all 180 LC fractions was depicted on the basis of their inhibitory effects on h CE2.The bioactive compounds displaying potent inhibitory effects towards h CE2 were identified by comparison of LC retention times,UV spectra and MS spectra,with the help of authentic standards.3.The inhibition behaviors of three identified natural h CE2 inhibitors were determined by using authentic standards.The IC50 values(concentration of inhibitor that reduces enzyme activity by 50%)were evaluated by nonlinear regression using Graph Pad Prism 6.0 software.The inhibition kinetic type was evaluated through determining the intersection point in the Dixon and Lineweaver-Burk plots.The second plot of slopes from Lineweaver-Burk plot versus inhibitor concentrations was utilized to calculate the corresponding inhibition parameter(Ki)value.4.The Calibration curves which were using to calculate the content of every bioactive compounds in WMR were determined by using authentic standards.Results: 1.Our results demonstrated that the ethanol extract of WMR displayed potent inhibitory effects against h CE2 with IC50 value of 30.32 μg/m L.2.Three bioactive compounds including Sanggenone D,Kuwanon G and Sanggenone C were efficiently identified by comparison of LC retention times,UV and MS spectral data,with the help of authentic standards.3.The inhibition potentials and inhibition types of these natural compounds against h CE2 were further investigated.The results demonstrated that these bioactive compounds are potent non-competitive inhibitors against h CE2,with the Ki values of 0.97 μM、1.09 μM、0.76 μM,respectively.4.The contents of SD,KG and SC were 576.5 μg/g dried bark materials,98.0 μg/g dried bark materials and 781.4 μg/g dried bark materials,respectively.Conclusion: All these findings implied that WMR and its major constitutes hold a good application prospect for alleviation of CPT-11 induced severe delayed-onset diarrhea.